Epithelial Na+ channels (ENaCs) are one of several key Na+ transporters in the aldosterone-sensitive distal nephron (ASDN) that participate in the reabsorption of filtered Na+ in a highly regulated manner. These Na+ transporters have important roles in the regulation of extracellular fluid volume and blood pressure. In addition to the ASDN, ENaCs are expressed at other sites that influence blood pressure. For example, ENaCs are expressed in antigen presenting dendritic cells where they appear to have a role in linking a high salt diet, inflammation and blood pressure. Proposed studies in Aim 1 will use a novel mouse ENaC gamma subunit hypomorph to elucidate the roles of ENaC in myeloid cells (dendritic cells) and non-myeloid cells (e.g., principal cells of the ASDN) in the blood pressure response to a high salt diet. Rare gain- or loss-of-function mutations within ENaC subunits have been described in Mendelian disorders characterized by hypertension or hypotension, respectively. Gene sequencing efforts have revealed hundreds of non-synonymous single nucleotide variants (nsNSVs) in human genes the encoding alpha, beta and gamma subunits. An increasing number of functional nsNSVs have been identified, although the potential contributions of the vast majority of these variants to human health and disease states are unknown. We have identified specific nsSNVs in the large extracellular regions of ENaC subunits that either enhance or inhibit channel activity by altering a regulatory response to extracellular Na+, referred to as Na+ self-inhibition. We have introduced specific gain- or loss-of-function variants into mice, corresponding to known functional human ENaC nsSNVs. Studies in Aim 2 will determine whether a gain-of-function variant predisposes mice to salt-sensitive hypertension, and whether a loss-of-function variant protects against salt-sensitive hypertension. Cleavage of ENaC subunits by specific proteases, and modification of ENaC subunits by the addition of palmitate, activate the channel. We have created novel mouse strains expressing ENaC subunits that lack key sites for protease cleavage or palmitoylation. Proposed studies in Aim 3 will define the roles of these regulatory processes in the adaptation to a low or high salt diet. The results from these studies will provide important insights regarding the role of dendritic cell ENaC in the blood pressure response to dietary salt in mice, demonstrate that ENaC variants that affect channel function by altering Na+ self-inhibition affect blood pressure in mice, and demonstrate the functional roles of ENaC subunit proteolysis and palmitoylation in mice.